This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) comprised of alternating glucuronic acid and N-acetylglucosamine units. These molecules are most widely recognized for their ability to bind proteins and modulate cellular events such as differentiation, blood coagulation, pathogenesis, and proliferation. Full understanding of these events requires knowledge of GAG structure. Although chip-based hydrophilic interaction chromatography (HILIC) is a powerful means of separating GAGs for on-line LC/MS, the negative-ion spray becomes unstable in high percent aqueous mobile phases. This work demonstrates the effectiveness of a chip designed with post-column addition of makeup LC flow to enable stable electrospray throughout the HILIC gradient. The result is a system with substantially increased spray stability and range of analytes that may be effectively analyzed. The mass spectrometric approach of analyzing heparan sulfate using CID has been limited by the dominant neutral loss of SO3 and consequently, the lack of glycosidic and cross-ring cleavage of the precursor ions. Since the loss of the sulfate is facilitated by mobile protons in the precursor ions, one possible solution would be to enhance the charge states during the ESI process to reduce available protons. Sulfolane and other agents have been shown to increase the charge states of protein complexes in the positive mode. In this study we demonstrate that sulfolane is able to increase both the ESI efficiency and charge states of HS oligosaccharide in the negative mode. We accomplish this by pulsing sulfolane via a novel post-column flow chip at designated time period during an online LC/MS